Reagent and method for phosphorus determination

ABSTRACT

A method and reagent for the determination of inorganic phosphate in biological fluids by reducing molybdiphosphate with a reducing agent, o-phenylenediamine dihydrochloride, in the presence of a catalyst, dimethylformamide; a preservative, thiourea; a nonionic surfactant stabilizer, and a polyvinylpyrrolidone clarifier, wherein significantly less acidity than heretofore is required.

United States Patent [191 Morin et al.

[ Dec. 10, 1974 1 REAGENT AND METHOD FOR PHOSPI-IORUS DETERMINATION [75] lnventors: Leo G. Morin; Jerome R. Prox, both of Miami, Fla.

[73] Assignee: Medico Electronic, Inc.,

Indianapolis, Ind.

[22] Filed: Aug. 30, 1972 [21] Appl. No.: 284,885

OTHER PUBLICATIONS R. J. Henry, Clinical Chemistry, Harper & Row, 1964,

N. D. Cheronis, Organic Chemistry, T. Y. Crowell C0., 1947, pp. 561-562.

M. M. Davis, National B ur. Standards Monograph, 105, 1968, pp. 17, 19.

General Aniline & Film Corp., Tech. Bull., 7543-066; 1960, pp. 1, 4.

Primary Examiner-Robert M. Reese Attorney, Agent, or FirmChristen & Sabol 5 7] ABSTRACT A method and reagent for the determination of inorganic phosphate in biological fluids by reducing molybdiphosphate with a reducing agent, 0- phenylenediamine dihydrochloride, in the presence of a catalyst, dimethylformamide; a preservative, thiourea; a nonionic surfactant stabilizer, and a polyvinylpyrrolidone clarifier, wherein significantly less acidity than heretofore is required.

30 Claims, No Drawings REAGENT AND METHOD FOR PHOSPHORUS DETERMINATION BACKGROUND OF THE INVENTION- 1. Field of this Invention This invention relates to a method for the determination of inorganic phosphate in biological fluids; and more particularly, this invention relates to a method for determining inorganic phosphate using phenylenediamine dihydrochloride to reduce molybdiphosphate in the presence of a dipolar aprotic solvent.

2. Prior Art The basis for the determination of inorganic phosphate is that acid molybdates react with phosphate to form heteropoly compounds of molybdiphosphate which are then reduced to molybdenum blue. The various methods in the literature for determining inorganic phosphates differ chiefly in the reducing agent employed. These reducing agents are well known in the art and they have been reviewed by Henry in Clinical Chemistry, (Harper & Row, 1968). Briefly, they are stannous chloride, phenylhydrazine, hydroquinone, ferrocyanide, ferrous sulfate, ammonium ferrous sulfate, ascorbic acid, aminonaphtholsulfonic acid, 2,4- diaminophenol, p-methylaminophenol sulfate, hydrazine, n-phenyl-p-phenylenediamine. All of these reduc' ing agents are not entirely satisfactory. The first three listed are characterized by unstable color, deviation from Beers Law, great sensitivity to change in acidity, and instability of the reducing agent itself. The next four obey Beers Law, but are slow acting and lack sensitivity. The remainder lack sensitivity. The remainder lack sensitivity, require high acidity (which results in elevated values due to hydrolysis of organic phosphates), and require elevated temperatures. All require at least 30 minutes. It is desirable to have a reducing agent that is stable, is fast acting, requiring no more than 5 minutes, sensitive, obeys Beers Law, requires no heating, requires low acidity, and produces stable color.

BROAD DESCRIPTION OF THE INVENTION It is a primary object of this invention to provide for I a significant improvement of the acid molybdate method for the determination of inorganic phosphate.

Another object of this invention is to provide a stable reducing agent. Still another object is to provide a sensitive reducing agent that produces intense color with a small sample.

Another object is to provide a reducing agent that requires no heat above ambient temperature. Still another object is to provide a reducing agent that acts rapidly, requiring no more than ten minutes. Another object is to'provide a reducing agent that obeys Beers law. Another object is to provide a reducing agent that requires only low acidity. Still another object is to provide a reducing agent that produces a stable color.

Another object of this invention is to provide a catalyst that accelerates the reaction so that the determination requires no more than five minutes.

Still another object is to provide a clarifier that will prevent turbidity in direct determinations.

Other objects and advantages of this invention will become more apparent from the following description.

Broadly, this invention involves an acid molybdate reagent for the determination of inorganic phosphate containing less than 30 percent sulfuric acid or its equivalent which is made to contain all or some of the following: o-phenylenediamine (and its salts), a dipolar aprotic solvent, thiourea, a nonionic surfactant, polyvinylpyrrolidone.

The term o-phenylenediamine includes 0- phenylenediamine and salts thereof such as the dihydrochloride. The preferred o-phenylenediamine is ophenylenediamine dihydrochloride.

This invention includes a reagent for determining inorganic phosphate in biological fluid which comprises an aqueous solution containing 0.5 to 30 percent of an acid, 0.1 to 2 gram percent of a molybdate salt, and ophenylenediarnine or a salt thereof. The reagent can also contain a dipolar aprotic solvent.

It has been found that o-phenylenediamine dihydrochloride is a superior reducing agent of molybdiphosphate to molybdynenum blue in that it is highly sensitive, fast, requires no heat, requires only low acidity, obeys Beers Law, and produces a stable color in less than ten minutes. From 10 mg percent to 1 gram percent of the reducing agent is used.

It has been further found that dipolar aprotic solvents act as catalysts to the reduction of molybdiphosphates to molybdenum blue regardless of the reducing agent used. From 1 to 5 percent of the dipolar aprotic solvent is used. The preferred dipolar aprotic solvent is dimethylformarnide.

It has been further determined that thiourea is a useful preservative and stabilizer of reducing agents used in the reduction of molybdiphosphate to molybdenum blue. The thiourea is present in an amount between 10 mg percent and 10 gram percent.

It has also been found that polyvinylpyrrolidone in conjunction with a nonionic surfactant or a mixture of nonionic surfactants acts as a useful clarifier in inorganic phosphate determination by the acid molybdate method. It has further been found that the nonionic surfactant, in addition to its clarifying properties, serves as a color stabilizer of molybdenum blue in the determination of inorganic phosphate by the acid molybdate method. The preferred nonionic surfactant 'is Triton X-l00. From 0.1 to 10 percent of a nonionic surfactant is used; and from 1 to 20 percent of polyvinylpyrrolidone.

The reagent in this invention can be prepared by dissolving 0.1 to 2 gram percent of ammonium molybdate (or any other molybdate salt that is phosphate-free) in 0.5 to 30 percent of sulfuric acid, or equivalent of another acid; adding 1 to 20 percent of polyvinylpyrrolidone (preferably its molecular weight is between 10,000 to 160,000), and l to 5 percent dimethylformamide, 0.01 to 10 percent Triton X-100, or its equivalent of another non-ionic surfactant; and finally, adding 10 mg percent to as much as 1 gram percent of ophenylenediamine dihydrochloride and 10 mg percent to 10 gram percent of thiourea.

This invention includes the method of determining the inorganic phosphate in biological fluid samples. Upon the addition of biological fluids in the ratio of l/100 to l/25, an intense stable blue color will develop in l to 10 minutes in linear proportion to the phosphate content of the sample. The phosphate concentration may be determined from the increase in absorbance between 500 to 800 nm by means of a colorimeter.

The biological fluids tested can be those of man or animal. Examples of biological fluids are serum, plasma, urine and spinal fluid.

This invention includes a reagent for determining inorganic phosphate in biological fluids which comprises an aqueous solution containing 1 to 5 percent of a dipolar aprotic solvent, 0.5 to 30 percent of an acid, 0.1 to 2 gram percent of a molybdate salt, and mg percent to 1 gram percent of a reducing agent. The reducing agent can be one of the novel reducing agents disclosed herein, such as, o-phenylenediamine dihydrochloride, or other o-phenylenediamine salts, or any suitable reducing agent such as those listed in the prior art section herein, such as stannous chloride, phenylhydrazine, hydroquinone, ferrocyandie, ferrous sulfate, ammonium ferrous sulfate, ascorbic acid, aminonaphtholsulfonic acid, 2,4-diaminophenol, p-methylaminophenol sulfate, hydrazine, and n-phenyl-p-phenylenediamine. The preferred dipolar aprotic solvent is dimethylformamide. This invention includes the method, along the lines described above, of colorimetrically determining the concentration of the inorganic phosphate in biological fluids by means of measuring the absorbance.

This invention includes a reagent for determining inorganic phosphate in biological fluids which comprises an aqueous solution containing 0.5 to 30 percent of an acid, 0.1 to 2 gram percent of a molybdate salt, 10 mg percent to 1 gram percent of a reducing agent, 0.01 to 10 percent of a nonionic surfactant, and 1 to percent of polyvinylpyrrolidone. The reducing agent can be the novel reducing agents disclosed herein or any suitable reducing agent such as those listed in the prior art section herein. The preferred surfactant is Triton X-100. This invention includes the method, along the lines described above, of colorimetrically determining the concentration of the inorganic phosphate in biological fluids by means of measuring the absorbance.

This invention includes a reagent for determining inorganic phosphate in biological fluids which comprises an aqueous solution containing 0.5 to 30 percent of an acid, 10 mg percent 1 gram percent of a reducing agent, 0.1 to 2 gram percent of a molybdate salt and 10 mg percent to 10 gram percent (preferably 5 gram percent) of thiourea. The reducing agent can be the novel reducing agents disclosed herein or any suitable reducing agent such as those listed in the prior art section herein. This invention includes the method, along the lines described above, of colorimetrically determining the concentration of the inorganic phosphate in biological fluids by means of measuring the absorbance.

DETAILED DESCRIPTION OF THE INVENTION The dipolar aprotic solvents act as catalysts for the reduction of molybdiphosphate to molybdenum blue regardless of the reducing agent used.

Monograph 105 of the National Bureau of Standards defines dipolar aprotic solvents. Aprotic solvents are almost devoid of acidic or basic properties, for example, aprotic solvents do not interact strongly with acidic solutes, such as carboxylic acids, phenols and mineral acids, or with basic solutes, such as amines and derivatives of quanidine or pyridine. They are comparatively inert in character. Aprotic solvents are not masking or leveling solvents, such as water. Indifferent solvents are other names for aprotic solvents. In general, aprotic solvents are organic compounds.

There are generally two classes of aprotic solvents, but this invention uses those aprotic solvents which are termed dipolar aprotic solvents (and which generally have a high dielectric constant (e) that ranges from about 21 to about 46.5). The preferred aprotic solvent is dimethylformamide. Examples of other aprotic solvents in this class are acetone, acetonitrile, nitrobenzene, nitromethane, methyl sulfoxide, benzonitrile, nitromethane, dimethylacetamide, sulfolane (tetramethylenesulfone), N-methyl formamide, formamide, N-methyl propionamide, dimethyl sulfoxide, diethylformamide, diethylacetimide, diethylsulfoxide, dipropylforrnamide, dipropylacetarnide, dipropylsulfoxide, dimethylpropiamide, dimethylbutyramide, and mixtures thereof.

The preferred molybdate salt is ammonium molybdate. Examples of useful molybdate salts are cadmium molybdate, cerium molybdate, barium molybdate, lanthanum molybdate, bismuth molybdate, lead molybdate, calcium molybdate, lithium molybdate, magnesium molybdate, potassium molybdate, sodium molybdate and zinc molybdate. The molybdate salt must be phosphate free. From 0.1 to 2 gram percent of molybdate salt is used.

The preferred acid is sulfuric acid, Examples of useful acids are hydrochloric acid, nitric acid, sulfurous acid, nitrous acid, bromoacetic acid, picric acid, bu tyric acid, salicylic acid, oxalic acid, formic acid, tartaric acid, lactic acid, maleic acid, acetic acid, citric acid, benzoic acid, and telluric acid. From 0.5 to 30 percent of acid is used.

The preferred nonionic surfactant is Triton X-lOO. Examples of other useful nonionic surfactants are the various polyoxyethylene sorbitan esters of fatty acids available under the tradename TWEEN and the nonylphenyl-polyethylene glycol esters available under the tradename BRIJ. From 0.1 to 10 percent of a nonionic surfactant can be used.

Any suitable colorimeter or spectrophotometer can be used to measure the absorbance. Examples of useful colorimeters are: Coleman, Model 44; Perkin-Elmer, Model 124; the colorimeter disclosed in US. Ser. No. 224,457, applicants: Raymond W. Kiess and Peter H. Stewart, filed: February 8, 1972, assignee: Kiess Instruments, Inc., 8768 S. W. 131st St., Miami, Fla., 33156; and the direct reading colorimeter disclosed in US. Pat. No. 3,561,878, inventor: R. W. Kiess.

The following examples further illustrate the invention but they do not limit it:

EXAMPLE 1 For convenience the reagent is prepared into three distinct components. Component A consists of 12 grams of ammonium molybdate brought to 1 liter with 24 percent sulfuric acid. Component B consists of 60 grams of polyvinylpyrrolidone (molecular weight of 40,000), 9.6 ml of dimethylformamide, and 1 ml of Triton X- brought to 1 liter with water. Component C consists of 30 grams of thiourea and 9 grams of ophenylenediarnine dihydrochloride brought to 100 ml with dimethylformamide. To a series of tubes is added 0.5 ml. of Component A, 2.5 ml of Component B, and 0.1 m1 of Component C to give the complete reagent.

EXAMPLE 2 The reagent is prepared as in Example 1 and reagent tubes are set up as in Example 1. A set of standard solutions are prepared in pooled human serum and made to contain 1,2, 3,4,5, 6,7, 8, 9 and mg percent of inorganic phosphate. To each tube of reagent is added 0.1 ml of human serum with various levels of phosphate. The results are the same as in Example 1.

EXAMPLE 3 The reagent is prepared as in Example 1, except that dimethylformamide is omitted and replaced with water. Standard solutions are tested as in Example 1. An intense stable blue color develops within 10 minutes instead of 5 minutes. The absorbance is found to be linear with the phosphate concentration.

EXAMPLE 4 The reagent is prepared as in Example 1, except that ascorbic acid is substituted for o-phenylenediamine dihydrochloride. The procedure is the same as in Example 1. An unstable blue color develops in minutes. The absorbance, although linear with the phosphate concentration, is of about 75 percent reduced intensity when compared with Example 1.

EXAMPLE 5 The reagent is prepared as in Example 4, except that dimethylformamide is omitted. The same procedure is followed as in Example 4. An unstable blue color develops in 30 minutes instead of 15 minutes. The absorbance is as in Example 4.

EXAMPLE 6 The reagent is prepared as in Example 1, except that p-methylaminophenol sulfate is substituted for ophenylenediamine dihydrochloride. The same procedure as in Example 1 is followed. No color develops.

EXAMPLE 7 The reagent is prepared as in Example 6, except that the sulfuric acid is increased from 24 percent to 75 percent. The procedure is the same as in Example 1. A weak green color develops in minutes that is linear with the phosphate concentration.

EXAMPLE 8 The reagent is prepared as in Example 1, except that the polyvinylpyrrolidone is omitted. The procedure is then the same as in 1. 1A turbid blue color develops that is unsuitable for measurement of absorbance.

EXAMPLE 9 The reagent is prepared as in Example 1 except that Triton -X-100 is omitted. The procedure is the same as in Example 1. A slightly turbid blue color develops that is linear with the phosphate concentration, but which is unstable and continues to darken indefinitely for at least 2 hours.

What is claimed is:

1. A reagent for determining inorganic phosphate in biological fluid which comprises an aqueous solution containing 0.5 to 30 percent of an acid, 0.1 to 2 gram percent of a molybdate salt, and o-phenylenediamine or a salt thereof.

2. A reagent according to claim 1 whrein the acid is sulfuric acid.

3. A reagent according to claim 1 wherein a dipolar aprotic solvent is present.

4. A reagent according to claim 3 wherein ophenylenediamine or salt thereof is present at a level between 10 mg percent to 1 gram percent.

5. A reagent according to claim 3 wherein the ophenylenediamine or salt thereof is ophenylenediarnine dihydrochloride.

6. A reagent according to claim 3 wherein the dipolar aprotic solvent is present at a level of l to 5 percent. I

7. A reagent according to claim 3 wherein the dipolar aprotic solvent is dimethylformamide,

8. A reagent according to claim 3 wherein thiourea is present at a level between 10 mg percent to 10 gram percent.

9. A reagent according to claim 3 wherein thiourea is present, wherein a nonionic surfactant is present, and wherein polyvinylpyrrolidone is present.

10. A reagent according to claim 3 wherein a nonionic surfactant is present in an amount between 0.01 to 10 percent.

1 l. A reagent according to claim 10 wherein the nonionic surfactant is Triton X-100.

12. A reagent according to claim 3 wherein polyvinylpyrrolidone is present at a level between 1 to 20 percent.

13. A reagent according to claim 12 wherein the polyvinylpyrrolidone has a molecular weight between 10,000 to 160,000.

14. A method for determining the inorganic phosphates in biological fluids which comprises admixing a sample of biological fluid with the reagent of claim 11, the ratio of the biological fluid to the reagent being between 1:100 and 1:25, and determining the concentration of inorganic phosphate by means of measuring the absorbance.

15. A method according to claim 14 wherein the ophenylenediarnine or salt thereof is present at a level between 10 mg percent to 1 gram percent.

16. A method according to claim 14 wherein the ophenylenediamine salt is o-phenylenediamine dihydrochloride.

17. A method according to claim 14 wherein thiourea is present, wherein a nonionic surfactant is present, and wherein polyvinylpyrrolidone is present.

18. A method according to claim 14 wherein the acid is sulfuric acid.

19. A method according to claim 14 wherein the absorbance is measured at a point between 500 and 800 20. A method according to claim 19 wherein thiourea is present at a level between 10 mg percent to 10 gram percent.

21. A method according to claim 19 wherein a nonionic surfactant is present in an amount between 0.1 to 10 percent.

22. A method according to claim 19 wherein polyvinylpyrrolidone is present at a level between 1 to 20 percent.

23. A method according to claim 19 wherein a dipolar aprotic is present at a level of l to percent.

24. A method according to claim 23 wherein the dipolar aprotic solvent is dimethylformamide.

25. A reagent for determining inorganic phosphate in biological fluids which comprises an aqueous solution containing 0.5 to 30 percent of an acid, mg percent to 1 gram percent of a reducing agent, 0.1 to 2 gram percent of a molybdate salt and 10 mg percent to 10 gram percent of thiourea.

26. A method for determining the inorganic phosphates in biological fluids which comprises admixing a sample of biological fluid with the reagent of claim 25, the ratio of the biological fluid to the reagent being between 11100 and 1:25, and determining the concentration of inorganic phosphate by means of measuring the absorbance.

27. A reagent for determining inorganic phosphate in biological fluids which comprises an aqueous soluton containing 0.5 to 30 percent of an acid, 0.1 to 2 gram percent of a molybdate salt, 10 mg percent to 1 gram percent of a reducing agent and l to 5 percent of a di polar aprotic solvent.

28. A method for determining the inorganic phosphates in biological fluids which comprises admixing a sample of biological fluid with the reagent of claim 27, the ratio of the biological fluid to the reagent being between 1:100 and 1:25, and determining the concentration of inorganic phosphate by means of measuring the absorbance.

29. A reagent for determining inorganic phosphate in biological fluids which comprises an aqueous solution containing 0.5 to 30 percent of an acid, 0.1 to 2 gram percent of a molybdate salt, 10 mg percent to 1 gram percent of a reducing agent, 0.0] to 10 percent of a nonionic surfactant, and 1 to 20 percent of polyvinylpyrrolidone.

30. A method for determining the inorganic phosphates in biological fluids which comprises admixing a sample of biological fluid with the reagent of claim 29, the ratio of the biological fluid to the reagent being between 1:100 and 1:25, and determining the concentration of inorganic phosphate by means of measuring the absorbance. 

1. A REAGENT FOR DETERMINING INORGANIC PHOSPHATE IN BIOLOGICAL FLUID WHICH COMPRISES AN AQUEOUS SOLUTION CONTAINING 0.5 TO 30 PERCENT OF AN ACID, 0.1 TO 2 GRAM PERCENT OF A MOLYBDATE SALT, AND O-PENHYLENEDIAMINE OR A SALT THEREOF.
 2. A reagent according to claim 1 whrein the acid is sulfuric acid.
 3. A reagent according to claim 1 wherein a dipolar aprotic solvent is present.
 4. A reagent according to claim 3 wherein o-phenylenediamine or salt thereof is present at a level between 10 mg percent to 1 gram percent.
 5. A reagent according to claim 3 wherein the o-phenylenediamine or salt thereof is o-phenylenediamine dihydrochloride.
 6. A reagent according to claim 3 wherein the dipolar aprotic solvent is present at a level of 1 to 5 percent.
 7. A reagent according to claim 3 wherein the dipolar aprotic solvent is dimethylformamide.
 8. A reagent according to claim 3 wherein thiourea is present at a level between 10 mg percent to 10 gram percent.
 9. A reagent according to claim 3 wherein thiourea is present, wherein a nonionic surfactant is present, and wherein polyvinylpyrrolidone is present.
 10. A reagent according to claim 3 wherein a nonionic surfactant is present in an amount between 0.01 to 10 percent.
 11. A reagent according to claim 10 wherein the nonionic surfactant is Triton X-100.
 12. A reagent according to claim 3 wherein polyvinylpyrrolidone is present at a level between 1 to 20 percent.
 13. A reagent according to claim 12 wherein the polyvinylpyrrolidone has a molecular weight between 10,000 to 160,000.
 14. A method for determining the inorganic phosphates in biological fluids which comprises admixing a sample of biological fluid with the reagent of claim 1, the ratio of the biological fluid to the reagent being between 1:100 and 1:25, and determining the concentration of inorganic phosphate by means of measuring the absorbance.
 15. A method according to claim 14 wherein the o-phenylenediamine or salt thereof is present at a level between 10 mg percent to 1 gram percent.
 16. A method according to claim 14 wherein the o-phenylenediamine salt is o-phenylenediamine dihydrochloride.
 17. A method according to claim 14 wherein thiourea is present, wherein a nonionic surfactant is present, and wherein polyvinylpyrrolidone is present.
 18. A method according to claim 14 wherein the acid is sulfuric acid.
 19. A method according to claim 14 wherein the absorbance is measured at a point between 500 and 800 nm.
 20. A method according to claim 19 wherein thiourea is present at a level between 10 mg percent to 10 gram percent.
 21. A method according to claim 19 wherein a nonionic surfactant is present in an amount between 0.1 to 10 percent.
 22. A method according to claim 19 wherein polyvinylpyrrolidone is present at a level between 1 to 20 percent.
 23. A method according to claim 19 wherein a dipolar aprotic is present at a level of 1 to 5 percent.
 24. A method according to claim 23 wherein the dipolar aprotic solvent is dimethylformamide.
 25. A reagent for determining inorganic phosphate in biological fluids which comprises an aqueous solution containing 0.5 to 30 percent of an acid, 10 mg percent to 1 gram percent of a reducing agent, 0.1 to 2 gram percent of a molybdate salt and 10 mg percent to 10 gram percent of thiourea.
 26. A method for determining the inorganic phosphates in biological fluids which comprises admixing a sample of biological fluid with the reagent of claim 25, the ratio of the biological fluid to the reagent being between 1:100 and 1:25, and determining the concentration of inorganic phosphate by means of measuring the absorbance.
 27. A reagent for determining inorganic phosphate in biological fluids which comprises an aqueous soluton containing 0.5 to 30 percent of an acid, 0.1 to 2 gram percent of a molybdate salt, 10 mg percent to 1 gram percent of a reducing agent and 1 to 5 percent of a dipolar aprotic solvent.
 28. A method for determining the inorganic phosphates in biological fluids which comprises admixing a sample of biological fluid with the reagent of claim 27, the ratio of the biological fluid to the reagent being between 1:100 and 1:25, and determining the concentration of inorganic phosphate by means of measuring the absorbance.
 29. A reagent for determining inorganic phosphate in biological fluids which comprises an aqueous solution containing 0.5 to 30 percent of an acid, 0.1 to 2 gram percent of a molybdate salt, 10 mg percent to 1 gram percent of a reducing agent, 0.01 to 10 percent of a nonionic surfactant, and 1 to 20 percent of polyvinylpyrrolidone.
 30. A method for determining the inorganic phosphates in biological fluids which comprises admixing a sample of biological fluid with the reagent of claim 29, the ratio of the biological fluid to the reagent being between 1:100 and 1:25, and determining the concentration of inorganic phosphate by means of measuring the absorbance. 